Tetrahydrobiopterin, a cofactor for aromatic amino acid hydroxylases, is associated with various diseases (Kaufman S & Fisher D B. (1974) Pterin-requiring aromatic amino acid hydroxylases. In: Hayaishi O, ed. Molecular Mechanisms of Oxygen Activation. New York: Academic Press pp 285-369).
Atypical phenylketonuria (PKU) is one of the representative diseases originated from tetrahydrobiopterin deficiency. An infant born having genetic deficiency in tetrahydrobiopterin biosynthesis or regeneration (that is an infant with PKU) shows dysfunction of phenylalanine hydroxylase, thereby increasing phenylalanine concentration in the blood (Nichol C A, Smith G K, Duch D S. (1985) Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 54:729764; Duch D S, Smith G K (1991) Biosynthesis and function of tetrahydrobiopterin. J. Nutr. Biochem., 2: 411-423). The patients suffering from PKU also show dysfunction in tyrosine and tryptophan hydroxylase activities, which results in insufficient biosynthesis of neurotransmitters such as dopamine and serotonin in the brain.
In addition, it has been reported that tetrahydrobiopterin deficiency causes dopa-responsive dystonia, which is one of the genetic neuronal diseases (Ichinose H, Ohye T, Takahashi E, et al. (1994) Hereditary progressive dystonia with marked diurnal fluctuation caused by mutation in the GTP cyclohydrolase I gene. Nat Genet 8:236-241). It has been also reported that tetrahydrobiopterin deficiency is associated with Parkinson's disease. And also, it has been reported that patients with Alzheimer's disease, depression, autism, or schizophrenia shows lower concentration of tetrahydrobiopterin in body fluids than normal people (Thony, B., Auerbach, G. and Blau, N. (2000) Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem. J. 347, 116; H. Tiemeier, D. Fekkes, A. Hofman, H. R. van Tuijl, A. J. Kiliaan, M. M. Breteler (2006) Plasma pterins and folate in late life depression: The Rotterdam study. Psychiatry Res. 145: 199-206; M. A. Richardson, L. L. Read, M. A. Reilly, J. D. Clelland, C. L. Clelland (2007) Analysis of plasma biopterin levels in psychiatric disorders suggests a common BH4 deficit in schizophrenia and schizoaffective disorder, Neurochem Res. 32: 107-113; T. Danfors, A. L. von Knorring, P, Hartvig, B. Langstrom, R. Moulder, B. Stromberg, R. Torstenson, U. Wester, Y. Watanabe, O. Eeg-Olofsson (2005) Tetrahydrobiopterin in the treatment of children with autistic disorder: a double-blind placebo-controlled crossover study, J. Clin. Psychopharmacol. 25: 485-489). Furthermore, vitiligo, which shows melanin deficiency in epidermis, is originated from dysfunction of tetrahydrobiopterin biosynthesis (Schallreuter K U, Zschiesche M & Moore J et al. (1998) In vivo evidence for compromised phenylalanine metabolism in vitiligo. Biochem Biophys Res Commun 243: 395-399).
Tetrahydrobiopterin also plays a critical role in endothelial dysfunctions such as diabetes, hypertension, myocardial infraction, and stroke, as a cofactor and regulator of nitric oxide synthase (NOS) (Schmidt T S, Alp N J (2007) Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease. Clinical Science 113: 47-63; and Moens A L, Kass D A (2006) Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol 26: 2439-2444).
In order to diagnose said various diseases such as atypical phenylketonuria, dystonia, Parkinson's disease, Alzheimer's disease, depression, autism, schizophrenia, vitiligo, and endothelial dysfunctions, it is required to effectively analyze tetrahydrobiopterin in a sample obtained from patients, e.g., blood (plasma and/or serum), cerebrospinal fluid, urine, and other tissues.
Meanwhile, tetrahydrobiopterin is functional in the fully reduced form (i.e., tetrahydro form). However, it is converted into non-functional oxidized forms, i.e., dihydrobiopterin and biopterin, under oxidative stress conditions, such as hypertension or diabetes. Therefore, both tetrahydrobiopterin and its oxidized forms (i.e., dihydrobiopterin and biopterin) may simultaneously exist in the body. Especially, in cardiovascular diseases which are known that the major cause thereof is oxidative stress, the oxidation of tetrahydrobiopterin is one of the serious problems. Recently, the ratio of tetrahydrobiopterin/(dihydrobiopterin+biopterin) or the ratio of tetrahydrobiopterin/(tetrahydrobiopterin+dihydrobiopterin+biopterin) is used as a key index in cardiovascular diseases (Yada T, Kaji S, Akasaka T, et al. (2007) Changes of asymmetric dimethylarginine, nitric oxide, tetrahydrobiopterin, and oxidative stress in patients with acute myocardial infarction by medical treatments. Clinical Hemorheology and Microcirculation 37: 269-276; and Taylor N E, Maier K G, Roman R J, Cowley A W (2006) NO synthase uncoupling in the kidney of Dahl rats: Role of dihydrobiopterin. Hypertension. 48: 1066-1071). Therefore, it is important to quantitatively analyze each amount of tetrahydrobiopterin and its oxidized forms in a biological sample.
Conventional analytical methods for tetrahydrobiopterin in a biological sample are based on fluorescence-characteristics of its oxidized forms. That is, tetrahydrobiopterin is oxidized with an acidic iodine solution and then quantitative analysis is performed using fluorometric high performance liquid chromatography (fluorometric HPLC). However, according to the conventional analytical methods, dihydrobiopterin and biopterin, in addition to tetrahydrobiopterin, are also oxidized and detected at the same position on HPLC. Therefore, the conventional analytical methods have a drawback that each tetrahydrobiopterin and its oxidized forms cannot be analyzed separately.
In order to solve the problem, Fukushima T et al. have developed an analytical method using alkaline iodine oxidation (Fukushima T, Nixon J C (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 102: 176-88). Under the condition of alkaline iodine oxidation, tetrahydrobiopterin is oxidized to pterin with cleavage of the side chain thereof; and dihydrobiopterin is oxidized to biopterin. However, the analytical method using alkaline iodine oxidation requires preparing a separate sample, in addition to a sample for acidic iodine oxidation, and also performing the HPLC analyses two times.
As another method, there has been reported a method, which includes performing HPLC in anaerobic conditions to separate tetrahydrobiopterin from its oxidized forms and then analyzing them with electrochemical detector (ECD) (Lunte C E, Kissinger P T (1983) The determination of pterins in biological samples by liquid chromatography/electrochemistry. Anal. Biochem 129: 377-386). However, the quantitative analysis using ECD shows significant deviations in each sample. In order to this problem, there has been also reported an improved method, wherein the samples eluted through the HPLC are oxidized with acidic iodine solution and then measured with a fluorometric detector (Hyland K (1985) Estimation of tetrahydro, dihydro and fully oxidized pterins by high performance liquid chromatography using sequential electrochemical and fluorometric detection. J Chromatogr. 343(1):3541). However, this method requires additional equipment for sample treatment.
Because of the above problems, it is difficult to quantitatively analyze tetrahydrobiopterin and its oxidized forms separately. And also, according to the literatures, significant deviations are shown in amount of tetrahydrobiopterin obtained from the same biological sample. Therefore, there is a need to develop a simple, prompt, and accurate method for quantitative analysis of each tetrahydrobiopterin and its oxidized forms in a sample, including biological samples.